Bottom Line:
Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix.The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

Affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.

ABSTRACTClostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4 Å resolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 Å resolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 Å resolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution, R = 16.9%, Rfree = 21.2%; 1.6 Å resolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

fig5: CαB-factor changes upon Ca2+ binding for s2a (a) and s2 (b). The CαB factors of holo s2b (c) are also shown. This figure was prepared using PyMOL (Schrödinger).

Mentions:
Overall, the average B factors of s2b and s2 are low and the average B factors of apo and holo s2a are similar (Table 1 ▶). Despite different symmetry interactions, the B factor per-residue trend of 18 PKD-like domains (eight holo s2a molecules, four apo s2a molecules, two holo s2b molecules and four apo s2 molecules) are very similar to each other (Supplementary Figs. S3 ▶a–S3d). Holo s2, however, does not follow this trend (Supplementary Fig. S3e). Comparison of holo and apo structures revealed a stark contrast between s2a and s2 in the influence of Ca2+ (Figs. 5 ▶a and 5 ▶c). Decreases in the CαB factor in holo s2 are found in three stretches (Gly698–Ile704, Gly708–Tyr721 and His738–Thr761) that are immediately preceded by stretches in which the B factor is higher (Lys691–Thr697, Glu705–Ser707 and Gly733–Val737). In the holo s2 structure the mid-section became more flexible, although both terminal regions became more rigid. Differences in crystal packing could account for the reversal in dynamics, although it is possible that the crystal packing is a consequence of dynamic changes. Both termini of holo s2 are pinned down by symmetry-related molecules that could suppress their dynamics, while the mid-section of the barrel lacks the intermolecular interactions observed in the apo state. In apo s2, intermolecular antiparallel β-sheet interactions involving β-strand A could suppress dynamics of the region.

fig5: CαB-factor changes upon Ca2+ binding for s2a (a) and s2 (b). The CαB factors of holo s2b (c) are also shown. This figure was prepared using PyMOL (Schrödinger).

Mentions:
Overall, the average B factors of s2b and s2 are low and the average B factors of apo and holo s2a are similar (Table 1 ▶). Despite different symmetry interactions, the B factor per-residue trend of 18 PKD-like domains (eight holo s2a molecules, four apo s2a molecules, two holo s2b molecules and four apo s2 molecules) are very similar to each other (Supplementary Figs. S3 ▶a–S3d). Holo s2, however, does not follow this trend (Supplementary Fig. S3e). Comparison of holo and apo structures revealed a stark contrast between s2a and s2 in the influence of Ca2+ (Figs. 5 ▶a and 5 ▶c). Decreases in the CαB factor in holo s2 are found in three stretches (Gly698–Ile704, Gly708–Tyr721 and His738–Thr761) that are immediately preceded by stretches in which the B factor is higher (Lys691–Thr697, Glu705–Ser707 and Gly733–Val737). In the holo s2 structure the mid-section became more flexible, although both terminal regions became more rigid. Differences in crystal packing could account for the reversal in dynamics, although it is possible that the crystal packing is a consequence of dynamic changes. Both termini of holo s2 are pinned down by symmetry-related molecules that could suppress their dynamics, while the mid-section of the barrel lacks the intermolecular interactions observed in the apo state. In apo s2, intermolecular antiparallel β-sheet interactions involving β-strand A could suppress dynamics of the region.

Bottom Line:
Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix.The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

Affiliation:
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.

ABSTRACTClostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4 Å resolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 Å resolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 Å resolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution, R = 16.9%, Rfree = 21.2%; 1.6 Å resolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.